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Comparison of the Effects on Mrna and Mirna Stability Arian Aryani and Bernd Denecke*

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Aryani and Denecke BMC Research Notes (2015) 8:164

DOI 10.1186/s13104-015-1114-z

  • RESEARCH ARTICLE
  • Open Access

In vitro application of ribonucleases: comparison of the effects on mRNA and miRNA stability

Arian Aryani and Bernd Denecke*

Abstract

Background: MicroRNA has become important in a wide range of research interests. Due to the increasing number of known microRNAs, these molecules are likely to be increasingly seen as a new class of biomarkers. This is driven by the fact that microRNAs are relatively stable when circulating in the plasma. Despite extensive analysis of mechanisms involved in microRNA processing, relatively little is known about the in vitro decay of microRNAs under defined conditions or about the relative stabilities of mRNAs and microRNAs. Methods: In this in vitro study, equal amounts of total RNA of identical RNA pools were treated with different ribonucleases under defined conditions. Degradation of total RNA was assessed using microfluidic analysis mainly based on ribosomal RNA. To evaluate the influence of the specific RNases on the different classes of RNA (ribosomal RNA, mRNA, miRNA) ribosomal RNA as well as a pattern of specific mRNAs and miRNAs was quantified using RT-qPCR assays. By comparison to the untreated control sample the ribonuclease-specific degradation grade depending on the RNA class was determined. Results: In the present in vitro study we have investigated the stabilities of mRNA and microRNA with respect to the influence of ribonucleases used in laboratory practice. Total RNA was treated with specific ribonucleases and the decay of different kinds of RNA was analysed by RT-qPCR and miniaturized gel electrophoresis. In addition, we have examined whether the integrity observed for ribosomal RNA is applicable to microRNA and mRNA. Depending on the kind of ribonuclease used, our results demonstrated a higher stability of microRNA relative to mRNA and a limitation of the relevance of ribosomal RNA integrity to the integrity of other RNA groups. Conclusion: Our results suggest that the degradation status of ribosomal RNA is not always applicable to mRNA and microRNA. In fact, the stabilities of these RNA classes to exposure to ribonucleases are independent from each other, with microRNA being more stable than mRNA. The relative stability of microRNAs supports their potential and further development as biomarkers in a range of applications.

Keywords: mRNA, miRNA, Ribosomal RNA, Ribonuclease, RNA stability, RNA integrity

Background

of their specific target genes [6]. The regulatory function of
MicroRNAs (miRNAs) are small, non-coding RNAs which miRNA is differentiated into two distinct mechanisms: the are encoded within the genome. The primary transcript, inhibition of target gene expression (i) by inducing the degpri-miRNA, contains one or more hairpin structures and is radation of the target mRNA [4] and/or (ii) by inhibition cleaved to pre-miRNA [1,2]. Upon export to the cytoplasm, of target gene translation [7]. Partial complementary base dicer cleaves the pre-miRNA to mature miRNA, which pairing causes translational inhibition whereas perfect base

  • usually has a length of about 22 ribonucleotides [1,3,4].
  • pairing causes degradation of the complementary target

Previous studies have suggested that mRNA stability is mRNAs. It has long been assumed that translational inhiban important control point for gene expression regulation ition is the dominant mechanism, however, recent studies [5]. The mature miRNA plays a key role in the regulation have shown that the degradation of target mRNA plays the major role in the inhibition of protein expression [8]. A widespread interest has developed in small non-coding RNAs for cancer research and drug development. In

* Correspondence: [email protected]

Interdisciplinary Center for Clinical Research Aachen (IZKF Aachen), RWTH Aachen University, Pauwelsstrasse 30, Aachen, Germany

© 2015 Aryani and Denecke; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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particular, miRNAs appear to play a critical role in tumori- constituent of total RNA in mammalian cells [15]. For this genesis and are thus promising candidates as potential reason the quality of mRNA is generally extrapolated on therapeutic targets or as biomarkers [9,10]. Moreover, miR- the basis of the quality of ribosomal RNA, which can be NAs are able to regulate the expression of multiple genes analysed, for example, with microfluidic analysis (Agilent and hence affect disease pathways at multiple points [10]. 2100 Bioanalyzer).

  • Therefore, miRNAs are promising tools in diagnostic and
  • Representative electropherograms of our samples that

therapeutic applications in a wide range of specializations, were analysed with the RNA6000 Nano labchip are including cancer research [11], cardiovascular disease [12], illustrated in Figure 1A. The electropherogram of the un-

  • organ transplantation [13] and rheumatoid arthritis [14].
  • treated sample consists of four peaks: i) the loading

Although miRNAs comprise an important class mole- marker at 25 nt, ii) the spiked tRNA at about 80 nt, iii) the cules used to uncover regulatory mechanisms in the cell, 18S ribosomal RNA and, iv) the 28S ribosomal RNA. The nearly nothing is known about their decay in comparison tRNA was added after the treatments in order to facilitate to mRNA under defined in vitro conditions. For this rea- the precipitation of RNA. It was clear that the 18S and son, we decided to enhance general knowledge about the 28S ribosomal RNAs were missing after NaOH treatment. stability of mRNA and miRNA in response to ribonucle- This was expected because NaOH, at the concentration ases commonly used in routine laboratory practice. We used, induces the hydrolysis of RNA in a manner that is were interested in finding out how a hypothetical contam- independent of size and sequence. Similar to NaOH, ination with ribonuclease might affect mRNA and miRNA RNase A-treated samples lacked both 18S and 28S peaks. stability. Furthermore, we investigated possible differences This indicated that both NaOH and RNase A treatments in the stability of different classes of RNA (i.e. miRNA, significantly affected ribosomal RNA.

  • mRNA or ribosomal RNA). For this purpose, total RNA
  • In the case of treatment with benzonase and RNase If,

was treated in vitro with a range of ribonucleases under even though the ribosomal peaks were not detectable by identical experimental conditions and their effects were this method, the measured absorption was above the

  • analysed by RT-qPCR.
  • base line. This is caused by residues of ribosomal RNA.

No specific changes in comparison to the untreated sample were observed in the electropherograms of the samples after treatment with RNase H, Exonuclease T or Exonuclease T7.

Results

The integrity of ribosomal RNA is affected by treatment with specific ribonucleases

  • In contrast to mRNA, which represents only 0.5 to 3% of
  • In order to determine the decay of ribosomal RNA

the transcriptome, ribosomal RNA is the most prevalent after the treatments with a higher sensitivity, a relative

Figure 1 The Stability of ribosomal RNA is affected in a treatment-dependent manner. (A) Representative electropherograms of untreated RNA (black) and RNA after treatment with either Benzonase (dark orange), NaOH (pink), RNase A (red), RNase H (grey), RNase If (bright orange), Exonuclease T7 (dark blue) or Exonuclease T (bright blue). Electropherogram of RNA ladder, used for size calculation indicated in the abscissa as kilobases (kb), is illustrated in green. Arbitrary units for fluorescence are given in the ordinate. The untreated control sample represents four peaks: the loading marker at 25 nt, the spiked tRNA at about 80 nt, the 18S and the 28S ribosomal RNA. (B) Detection of 18S ribosomal RNA using RT-qPCR. Percentage of intact 18S ribosomal RNA after indicated treatments is illustrated for three independent experiments with mean and SD (in red). The relative percentage of intact RNA in comparison to the untreated RNA is given in the ordinate. The green dotted line represents the 100% intact 18S ribosomal RNA obtained from the untreated RNA. Significances were calculated by the two tailed unpaired Student’s t-test: * = p < 0.05; *** = p < 0.001. ExoT7 = Exonuclease T7; ExoT = Exonuclease T.

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measurement of 18S RNA was performed with RT- Nppb (Cq = 19.8 0.8 cycles), Hprt (Cq = 24.7 0.4 cyqPCR (Figure 1B). The percentage of undigested 18S cles) and Polr2a (Cq = 26.9 0.5 cycles).

  • ribosomal RNA normalised to the untreated control is
  • On the basis of our previous quantitative studies [19],

presented in Figure 1B. It was observed that, similar to miR-1 and miR-208 (cardiac-specific expressed miRNAs) the results obtained by electrophoresis after treatment [20], sno202 (small nuclear RNA 202, often used as a with RNase A or NaOH, a significant reduction of ribo- normalization control) [21], and miR-501 [22] were insomal RNA was detectable (a reduction of 77% and 66%, vestigated as indicators of miRNA stability. Figure 2B ilrespectively). The observed difference in reduction be- lustrates the Cq values of the miRNA targets in the tween the two methods was probably caused by the untreated controls. Sno202 was more highly expressed

  • higher sensitivity of the RT-qPCR.
  • in heart tissue than the other miRNAs (Cq = 22.9

A similar effect could be observed after treatment with 0.5 cycles), followed by miR-1 (Cq = 23.3 0.3 cycles) benzonase and RNase If. In contrast to a dramatic re- and miR-208 (Cq = 25.8 0.2 cycles). For miR-501, the duction of ribosomal RNA detected by electrophoresis, lowest expression was detected (Cq =33.0 0.3 cycles). the results obtained by RT-qPCR showed only a moder- To determine whether normalization to a reference gene ate reduction. In the case of benzonase, this reduction influenced the results, we normalized the mRNA values was significant (p = 0.027). For treatment with RNase If, using Polr2, and the miRNA values using sno202. As ila statistically non-significant trend of reduction was lustrated in Figure 2C and D, the distribution of the

  • observed.
  • delta Cq values looked similar to the Cq values without

The treatments with RNase H, Exonucleases T and Exo- normalization. The relatively high standard deviation for nucleases T7 showed no significant changes compared to mRNA seems to be caused by the different expression the untreated sample. This confirmed the result obtained levels between male and females. Such a gender distinc-

  • by microfluidic analysis in a quantitative manner.
  • tion was not visible for the analyzed miRNAs.

  • In the case of RNase If treatment, comparison of sam-
  • For both mRNA and miRNA, the selected targets

ple electropherograms, which represented the 18S and cover a broad range of expression frequency (Cq 17.9 - 28S ribosomal RNAs (Figure 1A), with the results ob- 26.9 and Cq 22.9 - 33.0, respectively). This would ensure tained by 18S RT-qPCR (Figure 1B), revealed a discrep- the uncovering of potential treatment effects depending ancy between these two sets of results. The microfluidic on the frequency of a specific target RNA. However, a analysis revealed a partial degradation of 18S and 28S target RNA-dependent frequency was not observed. following treatment with RNase If , while the RT-qPCR Consequently, in order to compensate for the different demonstrated only a partial, non-significant digestion of target frequencies (i.e to have the result independent

  • the 18S ribosomal RNA.
  • from the original Cq value of each target), we normal-

Taken together, these results indicate that, under ex- ized the results to the Cq value of the corresponding unperimental conditions, RNase A, benzonase, NaOH and treated sample in our further analyses. RNase If treatments cause a total or partial decay of ribosomal RNA.

Ribonucleases do not have the same effect on different classes of RNA
Detection of mRNA and miRNA are represented by targets with different frequencies

Next we considered whether the stability of mRNA and miRNA was the same after the different treatments. We
Next we asked if the stability of ribosomal RNA to the measured the relative amount of intact mRNA/miRNA various treatments was representative of the stabilities of depending on the different treatments for the selected mRNA and/or miRNA to the same treatments. This targets. In Figure 3, the relative amount of the normalknowledge is necessary to assess the relative influence of ised intact targets is shown as a percentage of untreated various treatments on different classes of RNA. For this control. For every treatment, the percentages of intact reason we performed RT-qPCR for different mRNA and targets for three independent experiments are repremiRNA targets in the same samples as used for the riboso- sented for mRNA and miRNA. In addition, the sample

  • mal RNA analyses.
  • mean and standard deviation are shown. The results

The mRNA stability was investigated by RT-qPCR for could be divided into five categories. expression of Hprt (normally used as a reference gene) [16], Polr2a (normally used as a reference gene) [17], Nppa (gene expressed in heart muscles) and Nppb (gene also expressed in heart muscles) [18]. First, we compared the frequency of the targets analysed in the untreated samples. As illustrated in Figure 2A, Nppa was most highly expressed (Cq = 17.9 0.4 cycles), followed by

i) The first category represented the significant decay of mRNA and miRNA, including treatments with NaOH and RNase A. The RT-qPCR results for the mRNA targets suggested the total decay of mRNAs after the treatment with NaOH or RNase A. Similarly, after the NaOH treatment, the average of undigested

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Figure 2 Analysed targets represent a wide range of quantities. The quantification cycle (Cq), which is inversely proportional to the amount of target nucleic acid in the sample, was used to calculate the approximate amount of the targets. RT-qPCR analysis was performed with the untreated RNA isolated from three independent mice hearts (one male - grey, and two female - black). (A) Data-points and the mean of the Cq values of Nppa (cross), Nppb (downward-pointing triangle), Hprt (open circle) and Polr2a (hexagon) with the SD of three independent experiments are illustrated. (B) Data-points and the mean of the Cq values of Sno202 (closed circle), miR-1 (asterisk), miR-208 (rhombus) and miR-501 (upwardspointing triangle) with the SD of three independent experiments are illustrated. (C) Data-points and the mean of the ΔCq values of Nppa (cross), Nppb (downward-pointing triangle), Hprt (open circle) and Polr2a (hexagon) with the SD of three independent experiments after normalization with the reference mRNA Polr2. (D) Data-points and the mean of the ΔCq values of Sno202 (upwards-pointing triangle), miR-1 (closed circle), miR-208 (asterisk) and miR-501 (rhombus) with the SD of three independent experiments after normalization with the reference small RNA sno202.

miRNAs was reduced by 48.5%. The reduction for RNase A-treated samples was more prominent (i.e. 87.9%). Therefore, these treatments resulted in a severe decay of miRNAs. These results are consistent with those obtained by microfluidic analysis and by RT-qPCR for the

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could also be observed in the electropherogram of the microfluidic analysis. However, the partial digestion of 18S RNA represented by RT-qPCR was not significant (Figure 1B). iv) RNase H represented the fourth category, which caused no effect on mRNA or miRNA or ribosomal RNA. v) Benzonase represented the last category. After treatment with benzonase, a significant reduction of intact mRNAs was observed (20.4%). Contrary to this result, no reduction was seen for miRNA. The partial digestion of ribosomal RNA by benzonase as analysed by microfluidic analysis and 18S RT-qPCR was in line with the results of those for mRNA. Thus, benzonase precisely affected ribosomal RNA and mRNA, but had no effect on miRNA.

The results of our study indicate that, depending on the kind of treatment (i.e. ribonucleases), mRNA and miRNA demonstrate different stabilities. Notably, in comparison to mRNA, miRNA frequently presented a higher level of stability. Taken together, our results indicate that NaOH and all analysed ribonucleases (except RNase H) affected mRNA and resulted in the decay of mRNA. On the one hand, benzonase, Exonuclease T7 and Exonuclease T significantly affected mRNA but had no significant effect on decay of miRNA. On the other hand, treatments with NaOH, RNase A and RNase If also affected miRNA. In addition, after specific treatments of total RNA, microfluidic analyses revealed that ribosomal RNA was degraded whereas miRNA, as assessed by RT-qPCR, exhibited no degradation relative to the untreated sample. Hence, we conclude that there is not always a direct correlation between the integrity of ribosomal RNA and that of mRNA or of miRNA.

Figure 3 mRNA and miRNA stabilities measured by RT-qPCR are differently influenced depending on the treatment. Percentage of intact mRNA (blue) and miRNA (black) after indicated treatments are illustrated for three independent experiments. Nppa, Nppb, Hprt, and Polr2a were each analysed as targets representative for mRNA and sno202, miR1, miR208, and miR501 each as targets for miRNA. The mean with the SD for all analysed targets of each RNA class is given in violet for mRNA and in red for miRNA. The green dotted line represents the 100% intact RNA obtained from the untreated RNA. The ordinate indicates the relative percentage of intact RNA in comparison to the untreated RNA. Significances were tested by the two tailed unpaired Student’s t-test: * = p < 0.05; ** = p < 0.01; *** = p < 0.001. ExoT7 = Exonuclease T7; ExoT = Exonuclease T.

Discussion

Significant amounts of miRNAs, which are remarkably stable, can be detected in all biological fluids. This has favoured the use of miRNAs as a new class of biomarkers. It is assumed that the high stability of miRNAs in biological fluids is based on the fact that they are protected from endogenous RNase activity by being packed inside exosomes or that they are protected via association with an AGO protein complex [23,24]. The purpose of the present study was to investigate the stability of different classes of RNA (miRNA, mRNA, and rRNA) against common sources of contamination in a molecular biology laboratory. In eukaryotes, the most common method used to assess the integrity of RNA is the measurement of the 28S to 18S ribosomal RNA where the ratio for intact RNA is 2:1. Previously, this assessment was done by running an RNA aliquot on a denaturing agarose gel stained with

ribosomal RNA, which also showed a significant amount of decay. ii) The second category consisted of Exonuclease T and
Exonuclease T7 treatments which resulted in the digestion of mRNAs but not of miRNAs. Although the reduction of undigested mRNA was only by about 2% in both cases, it was found to be significant. As in the case of miRNA, ribosomal RNA did not show any changes in RNA integrity or in 18S levels by RT-qPCR (Figure 1). iii)The third category was represented only by RNase
If. The amounts of intact mRNA and miRNA were both moderately, but significantly reduced (11.9% for mRNA and 6.6% for miRNA). This indicated that RNase If digested miRNA as well as mRNA, but in a moderate manner. Digestion of ribosomal RNA

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ethidium bromide. Other nucleic acid stains such as biology laboratory. These ribonucleases are frequently SYBR Gold increase the sensitivity, also enabling the de- connected with a high possibility of reaction contamintection of lower amounts loaded on the gel. In the ation. To our knowledge, up to the time of this study, meantime, RNA integrity is assessed using microfluidic there is no information available regarding the in vitro analysis (e.g. Agilent 2100 Bioanalyzer, AdvanceCE™ Nu- effect of the analysed ribonucleases on classes of RNA.

  • cleic Acids Analyzer, or BioRad Experion). By contrast,
  • In a previous study it was shown that, even in pre-

absorbance or fluorescent dye-based procedures are use- sumed heat-degraded total RNA, the robust stability of ful for the quantification of RNA and, if necessary, for miRNA enabled its quantification. In contrast, the measthe purity. These techniques are, however, unsuitable for urement was no longer possible for mRNA [27]. This the determination of RNA integrity. The assessment of study has also shown that the measured RNA integrity mRNA or miRNA integrity in total RNA, independent does not adversely affect the accuracy of RT-qPCR meaof ribosomal RNA, is proving extremely difficult and surements of miRNA, although it significantly affects mostly includes RT-qPCR. Although there are also the measurement of mRNA [27]. Thus, degradation by microfluidic analysis assays for small RNAs (e.g. Agilent heat results in major effects on mRNA in comparison to small RNA assay) these assays are influenced by total miRNA, and miRNA represents a relatively stable form. RNA integrity [25]. For these reasons, microfluidic ana- Another study also indicated that miRNA possess dislysis of total RNA is widely used for the assessment of tinct stability. This stability depends on factors such as 7 total RNA integrity. The integrity of mRNA and miRNA nucleotides on 3′ terminal or the specific sequences is normally extrapolated from the measured ribosomal within a miRNA that make the miRNA a target for ribo-

  • integrity.
  • nucleases. Also, some miRNAs are associated with com-

Our results show that the degradation status of ribo- ponents of RISC such as Ago 2, which also influence the somal RNA is not, in all cases, applicable for the deg- miRNA stability [28]. However, in this study we were radation status of mRNA and miRNA. In addition, a more interested in comparing the stability of mRNA and class-dependent stability for mRNA, miRNA and ribo- miRNA after treatment with commonly used ribonuclesomal RNA was detected in presence of specific ribo- ases, being the main source of contamination in routine nucleases. The class-dependent stability was supported molecular biology laboratories. This important aspect by the observation that the analysed - RNA class- may affect the assessment of RNA quality and thus subspecific - targets showed, more or less, the same stabil- sequent results of experiments. Precisely, we have shown ity. In general, miRNAs revealed more stability than that degradation by different ribonucleases is RNA classmRNAs to exposure to ribonucleases and NaOH. This dependent. Our results also confirmed that RNA integ-

  • indicates miRNAs to be good, candidate biomarkers.
  • rity would not consistently affect the assessment of the

Apart from the important regulatory effect of miRNA degradation of miRNA.

  • on mRNA stability and/or translation [8], the dysregula-
  • RNase A is an endoribonuclease that attacks single-

tion of miRNAs can function as a biomarker for use in stranded RNA 3′ to pyrimidine residues and cleaves the cancer diagnosis and for many other diseases [10-14]. In phosphate linkage to the adjacent nucleotide [29,30]. this context it is important to know if the observed dys- This RNase is one of the major contamination sources regulation is due to the regulation process or due to the in the molecular biology laboratory. NaOH also catalyses

  • differential stabilities of mRNA and miRNA.
  • the breaking of the phosphate backbone in RNA. As ex-

Differential stabilities may have extensive consequences pected, both treatments (RNase A and NaOH) signififor analysis of the observed data. For instance, in the case cantly decayed all three analysed classes of RNA.

  • of detection of increased levels of miRNA and a decreased
  • Moreover, RNase If is one of the few known ribonucle-

level of the corresponding target(s) mRNA, the conclusion ases which cleaves the phosphodiester bond between any would be that the putative down-regulation of mRNA is two nucleotides [31]. It is a single strand specific endoridue to the regulatory effect of miRNA. However, the lower bonuclease. It was reported that RNase If favours the stability of mRNA would not have been considered. Nor- degradation of small RNA oligonucleotides but also demalization to endogenous reference genes is a useful grades long RNA polymers very slowly [32]. This earlier procedure to correct for sample-to-sample variations in finding would explain why all three examined classes of RT-qPCR efficiency and errors in sample quantification. RNA decayed, however, the observable decay for ribosoThe expression is estimated relative to the levels of the mal RNA was not found to be significant.

  • (invariant!) endogenous references [26]. However, this correct-
  • RNase H cleaves RNA via a hydrolytic mechanism in

ive action does not take effect between mRNA and miRNA, DNA/RNA duplexes [33]. This was confirmed in our experespecially if these are degraded with different efficiencies. Furthermore, we have performed the experiments with iments because no decay was observed in any class of RNA. It was reported that Exonuclease T7 functions similar ribonuclesases that are commonly used in a molecular to RNase H on double-stranded DNA/RNA hybrids. It

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    G C A T T A C G G C A T genes Review RNA Epigenetics: Fine-Tuning Chromatin Plasticity and Transcriptional Regulation, and the Implications in Human Diseases Amber Willbanks, Shaun Wood and Jason X. Cheng * Department of Pathology, Hematopathology Section, University of Chicago, Chicago, IL 60637, USA; [email protected] (A.W.); [email protected] (S.W.) * Correspondence: [email protected] Abstract: Chromatin structure plays an essential role in eukaryotic gene expression and cell identity. Traditionally, DNA and histone modifications have been the focus of chromatin regulation; however, recent molecular and imaging studies have revealed an intimate connection between RNA epigenetics and chromatin structure. Accumulating evidence suggests that RNA serves as the interplay between chromatin and the transcription and splicing machineries within the cell. Additionally, epigenetic modifications of nascent RNAs fine-tune these interactions to regulate gene expression at the co- and post-transcriptional levels in normal cell development and human diseases. This review will provide an overview of recent advances in the emerging field of RNA epigenetics, specifically the role of RNA modifications and RNA modifying proteins in chromatin remodeling, transcription activation and RNA processing, as well as translational implications in human diseases. Keywords: 5’ cap (5’ cap); 7-methylguanosine (m7G); R-loops; N6-methyladenosine (m6A); RNA editing; A-to-I; C-to-U; 2’-O-methylation (Nm); 5-methylcytosine (m5C); NOL1/NOP2/sun domain Citation: Willbanks, A.; Wood, S.; (NSUN); MYC Cheng, J.X. RNA Epigenetics: Fine-Tuning Chromatin Plasticity and Transcriptional Regulation, and the Implications in Human Diseases. Genes 2021, 12, 627.
  • SARS-Cov-2 RNA, Qualitative Real-Time RT-PCR (Test Code 39433)

    SARS-Cov-2 RNA, Qualitative Real-Time RT-PCR (Test Code 39433)

    SARS-CoV-2 RNA, Qualitative Real-Time RT-PCR (Test Code 39433) Package Insert For Emergency Use Only For In-vitro Diagnostic Use - Rx Only Intended Use The Quest Diagnostics SARS-CoV-2 RNA, Qualitative Real-Time RT-PCR (“Quest SARS-CoV-2 rRT-PCR”) is a real-time RT-PCR test intended for the qualitative detection of nucleic acid from the SARS-CoV-2 in upper and lower respiratory specimens (such as nasopharyngeal or oropharyngeal swabs, sputum, tracheal aspirates, and bronchoalveolar lavage) collected from individuals suspected of COVID-19 by their healthcare provider. This test is also for use with nasal swab specimens that are self-collected at home or in a healthcare setting by individuals using an authorized home-collection kit when determined to be appropriate by a healthcare provider. This test is for the qualitative detection of nucleic acid from the SARS-CoV-2 in pooled samples containing up to four of the individual upper respiratory swab specimens (nasopharyngeal, mid-turbinate, anterior nares or oropharyngeal swabs) that were collected in individual vials containing transport media from individuals suspected of COVID-19 by their healthcare provider. Negative results from pooled testing should not be treated as definitive. If patient’s clinical signs and symptoms are inconsistent with a negative result or results are necessary for patient management, then the patient should be considered for individual testing. Specimens included in pools with a positive, inconclusive, or invalid result must be tested individually prior to reporting a result. Specimens with low viral loads may not be detected in sample pools due to the decreased sensitivity of pooled testing.
  • U6 Small Nuclear RNA Is Transcribed by RNA Polymerase III (Cloned Human U6 Gene/"TATA Box"/Intragenic Promoter/A-Amanitin/La Antigen) GARY R

    U6 Small Nuclear RNA Is Transcribed by RNA Polymerase III (Cloned Human U6 Gene/"TATA Box"/Intragenic Promoter/A-Amanitin/La Antigen) GARY R

    Proc. Nati. Acad. Sci. USA Vol. 83, pp. 8575-8579, November 1986 Biochemistry U6 small nuclear RNA is transcribed by RNA polymerase III (cloned human U6 gene/"TATA box"/intragenic promoter/a-amanitin/La antigen) GARY R. KUNKEL*, ROBIN L. MASERt, JAMES P. CALVETt, AND THORU PEDERSON* *Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545; and tDepartment of Biochemistry, University of Kansas Medical Center, Kansas City, KS 66103 Communicated by Aaron J. Shatkin, August 7, 1986 ABSTRACT A DNA fragment homologous to U6 small 4A (20) was screened with a '251-labeled U6 RNA probe (21, nuclear RNA was isolated from a human genomic library and 22) using a modified in situ plaque hybridization protocol sequenced. The immediate 5'-flanking region of the U6 DNA (23). One of several positive clones was plaque-purified and clone had significant homology with a potential mouse U6 gene, subsequently shown by restriction mapping to contain a including a "TATA box" at a position 26-29 nucleotides 12-kilobase-pair (kbp) insert. A 3.7-kbp EcoRI fragment upstream from the transcription start site. Although this containing U6-hybridizing sequences was subcloned into sequence element is characteristic of RNA polymerase II pBR322 for further restriction mapping. An 800-base-pair promoters, the U6 gene also contained a polymerase III "box (bp) DNA fragment containing U6 homologous sequences A" intragenic control region and a typical run of five thymines was excised using Ava I and inserted into the Sma I site of at the 3' terminus (noncoding strand). The human U6 DNA M13mp8 replicative form DNA (M13/U6) (24).
  • The Difficult Case of an RNA-Only Origin of Life

    The Difficult Case of an RNA-Only Origin of Life

    Emerging Topics in Life Sciences (2019) 3 469–475 https://doi.org/10.1042/ETLS20190024 Perspective The difficult case of an RNA-only origin of life Kristian Le Vay and Hannes Mutschler Max-Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany Downloaded from https://portlandpress.com/emergtoplifesci/article-pdf/3/5/469/859756/etls-2019-0024c.pdf by Max-Planck-Institut fur Biochemie user on 28 November 2019 Correspondence: Hannes Mutschler ([email protected]) The RNA world hypothesis is probably the most extensively studied model for the emergence of life on Earth. Despite a large body of evidence supporting the idea that RNA is capable of kick-starting autocatalytic self-replication and thus initiating the emergence of life, seemingly insurmountable weaknesses in the theory have also been highlighted. These problems could be overcome by novel experimental approaches, including out-of- equilibrium environments, and the exploration of an early co-evolution of RNA and other key biomolecules such as peptides and DNA, which might be necessary to mitigate the shortcomings of RNA-only systems. The conjecture that life on Earth evolved from an ‘RNA World’ remains one of the most popular hypotheses for abiogenesis, even 60 years after Alex Rich first put the idea forward [1]. For some, evi- dence based upon ubiquitous molecular fossils and the elegance of the idea that RNA once had a dual role as information carrier and prebiotic catalyst provide overwhelming support for the theory. Nevertheless, doubts remain surrounding the chemical evolution of an RNA world, whose classical scenario is based on a temporal sequence of nucleotide formation, enzyme-free polymerisation/replica- tion, recombination, encapsulation in lipid vesicles (or other compartments), evolution of ribozymes and finally the innovation of the genetic code and its translation (Figure 1)[2,3].
  • Expanding the Genetic Code Lei Wang and Peter G

    Expanding the Genetic Code Lei Wang and Peter G

    Reviews P. G. Schultz and L. Wang Protein Science Expanding the Genetic Code Lei Wang and Peter G. Schultz* Keywords: amino acids · genetic code · protein chemistry Angewandte Chemie 34 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/anie.200460627 Angew. Chem. Int. Ed. 2005, 44,34–66 Angewandte Protein Science Chemie Although chemists can synthesize virtually any small organic molecule, our From the Contents ability to rationally manipulate the structures of proteins is quite limited, despite their involvement in virtually every life process. For most proteins, 1. Introduction 35 modifications are largely restricted to substitutions among the common 20 2. Chemical Approaches 35 amino acids. Herein we describe recent advances that make it possible to add new building blocks to the genetic codes of both prokaryotic and 3. In Vitro Biosynthetic eukaryotic organisms. Over 30 novel amino acids have been genetically Approaches to Protein encoded in response to unique triplet and quadruplet codons including Mutagenesis 39 fluorescent, photoreactive, and redox-active amino acids, glycosylated 4. In Vivo Protein amino acids, and amino acids with keto, azido, acetylenic, and heavy-atom- Mutagenesis 43 containing side chains. By removing the limitations imposed by the existing 20 amino acid code, it should be possible to generate proteins and perhaps 5. An Expanded Code 46 entire organisms with new or enhanced properties. 6. Outlook 61 1. Introduction The genetic codes of all known organisms specify the same functional roles to amino acid residues in proteins. Selectivity 20 amino acid building blocks. These building blocks contain a depends on the number and reactivity (dependent on both limited number of functional groups including carboxylic steric and electronic factors) of a particular amino acid side acids and amides, a thiol and thiol ether, alcohols, basic chain.
  • The Coffee Protective Effect on Catalase System in the Preneoplastic Induced Rat Liver

    The Coffee Protective Effect on Catalase System in the Preneoplastic Induced Rat Liver

    Hindawi Publishing Corporation Journal of Chemistry Volume 2016, Article ID 8570321, 9 pages http://dx.doi.org/10.1155/2016/8570321 Research Article The Coffee Protective Effect on Catalase System in the Preneoplastic Induced Rat Liver Cristiana Schmidt de Magalhães,1 Jéssica Emi Takarada,1 Nathália Costa Carvalho,1 Dayene do C. Carvalho,2 Felipe Lopes de Andrade,1 Eric Batista Ferreira,1 Pedro Orival Luccas,2 and Luciana Azevedo3 1 Exact Sciences Institute, Federal University of Alfenas, Rua Gabriel Monteiro da Silva 700, Centro, 37130-000 Alfenas, MG, Brazil 2Chemistry Institute, Federal University of Alfenas, Rua Gabriel Monteiro da Silva 700, Centro, 37130-000 Alfenas, MG, Brazil 3Nutrition Faculty, Federal University of Alfenas, Rua Gabriel Monteiro da Silva 700, Centro, 37130-000 Alfenas, MG, Brazil Correspondence should be addressed to Cristiana Schmidt de Magalhaes;˜ [email protected] Received 15 October 2015; Revised 2 March 2016; Accepted 28 March 2016 Academic Editor: Philippe Jeandet Copyright © 2016 Cristiana Schmidt de Magalhaes˜ et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This study aimed to evaluate the effect of organic/conventional coffee in liver tissues in the cancer process, taking into account the level and activities of catalase. The experiments were carried out with 8 groups of rats during 12 weeks. They received two injections of ethylenediaminetetraacetic acid solution 1.5% (v/v) prepared in 0.9% NaCl or 1,2-dimethylhydrazine (DMH) subcutaneous dose −1 −1 of 40 mg⋅kg ⋅bw for 2 weeks.
  • RNA Quality Control in Mirna Expression Analysis

    RNA Quality Control in Mirna Expression Analysis

    RNA Quality Control in miRNA Expression Analysis Application Note Genomics Authors Abstract Christiane Becker, Michael W. Pfaffl It is generally known that total RNA quality has a distinct influence on the validity and TU München, ZIEL reliability of quantitative PCR results. In addition, the recently published MIQE Physiology Weihenstephan guidelines focus on the pre-PCR steps and state the importance of RNA quality Weihenstephaner Berg 3 assessment. 85384 Freising, Germany Various studies showed the impairing effect of ongoing RNA degradation on mRNA Martina Reiter, Michael W. Pfaffl expression results. Therefore, the verification of RNA integrity prior to downstream BioEPS GmbH, Lise-Meitner-Str. 30 applications like RT-qPCR and mircroarrays is indispensable. A fast and reliable 85354 Freising, Germany assessment of RNA integrity can be done with the Eukaryote Total RNA Nano Assay of the Agilent 2100 Bioanalyzer System. The importance of RNA quality should also be considered in new applications such as the investigation of miRNA expression profiles. With the Agilent Small RNA Assay, Agilent is offering one of the few possibilities for selectively estimating miRNA before expression analysis. However, by now little is known about factors affecting miRNA analysis. Herein, the important impact of total RNA quality on quantification of mRNA and miRNA should be considered. Introduction After extraction, RNAs are very unsta- ble and sensitive to degradation due to 1A the ubiquitous occurrence of nucleases. It is well known that expression profil- ing using microarrays or qPCR is influ- enced by RNA quality1, 2. Therefore, this hazard has to be considered during all preprocessing steps and should be min- imized with cautious handling.
  • „Transformation Von Phospholipiden Durch Phospholipasen A1 Und

    „Transformation Von Phospholipiden Durch Phospholipasen A1 Und

    „Transformation von Phospholipiden durch Phospholipasen A1 und Phospholipasen D“ Kumulative Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) vorgelegt der Naturwissenschaftlichen Fakultät I Biowissenschaften der Martin-Luther-Universität Halle-Wittenberg von Martin Dippe geboren am 3. August 1981 in Wernigerode (Harz) Gutachter: 1. Prof. Renate Ulbrich-Hofmann (Martin-Luther-Universität Halle-Wittenberg) 2. Prof. Ingo Heilmann (Martin-Luther-Universität Halle-Wittenberg) 3. Prof. Uwe T. Bornscheuer (Ernst-Moritz-Arndt-Universität Greifswald) Halle (Saale), den 08. Dezember 2011 Inhaltsverzeichnis Inhaltsverzeichnis Inhaltsverzeichnis .............................................................................................................. 1 Abkürzungsverzeichnis ..................................................................................................... 2 1 Einleitung und Zielstellung .......................................................................................... 3 2 Theoretischer Teil ....................................................................................................... 5 2.1. Phospholipide – Molekularer Bau, Verwendung und chemische Synthese .... 6 2.2. Phospholipase-vermittelte Modifizierung von Phospholipiden ....................... 8 2.3. PLD-Enzyme............................................................................................... 10 2.3.1. Funktion und Mechanismus ............................................................ 10 2.3.2. Kopfgruppenaustausch